The invention relates to calorimeters, e.g., microcalorimeters, and calorimetry methods.
Microcalorimeters are devices that measure very small quantities of heat. In chemistry, biochemistry, cell biology, and pharmacology, ultrasensitive microcalorimeters are frequently used to measure thermodynamic properties of biological macromolecules, such as proteins.
A typical microcalorimeter has two cells, a xe2x80x9creference cellxe2x80x9d filled with a liquid, and a xe2x80x9csample cellxe2x80x9d filled with a dilute solution of a test substance, e.g., a macromolecule, in the same liquid. By comparing the heat evolved or absorbed by the two cells in response to changes in certain stimuli, such as temperature or concentration of a ligand, information about the test substance can be determined.
There are generally two popular types of microcalorimeters: the differential scanning calorimeter and the isothermal titration calorimeter. The differential scanning calorimeter automatically raises or lowers the temperature of the system at a given rate, while monitoring any temperature differential that arises between the two cells. From the temperature differential information, small differences between the amount of heat absorbed or released by the sample cell in comparison to the reference cell can be determined and attributed to the test substance.
In isothermal titration calorimetry, the instrument maintains a constant temperature while the concentration of an additional substance added to the cells is varied. The additional substance can be, e.g., a ligand that binds to the test substance in the sample cell. The instrument measures the heat absorbed or released as the newly introduced ligand binds to the test substance. By repeating the titration experiment using multiple additions of the ligand until binding is complete, various information concerning the interaction between the test substance and the ligand, e.g., stoichiometry, binding constant, and heat of binding, can be determined.
In general, in one aspect, the invention features a calorimeter that includes a sample cell, a reference cell, a pressure system that applies a variable pressure to the sample cell, and a pressure controller that controls the pressure applied by the pressure system to the sample cell.
Embodiments of this aspect of the invention may include one or more of the following features. The pressure system can apply the variable pressure to both the sample cell and the reference cell simultaneously. For example, the pressure system can be a pressure chamber that communicates continuously with both the sample cell and the reference cell. The pressure controller can include first and second pressure sources that apply first and second pressures, respectively, to the pressure chamber, and a control valve can be used to regulate whether the first or second pressure is applied to the chamber.
The calorimeter can further include a heat monitoring system that determines differences between the amount of heat absorbed or released by the sample cell and by the reference cell. The heat monitoring system can include a temperature sensor that monitors a temperature differential between the two cells that arises, e.g., in response to a change in the pressure applied by the pressure system.
The sample cell can be a vessel shaped to contain a liquid, and the pressure system can apply the variable pressure to a liquid holding portion of the sample cell. The reference cell can be substantially identical in mass and volume to the sample cell.
The reference cell can contain a liquid, and the sample cell can contain a solution that includes the liquid and a test substance, such as a biopolymer.
The calorimeter can also have an electrical control system electrically coupled to the pressure controller. The electrical control system includes, for example, a computer program that causes the pressure controller to periodically vary the pressure applied by the pressure system. In addition, the calorimeter can include a heating assembly thermally coupled to the sample and reference cells and electrically coupled to the control system. The same computer program can also cause the heating assembly to change the temperature of the sample and reference cells at a rate specified by a user, and can cause the pressure controller to periodically vary the pressure applied by the pressure system.
The calorimeter can also have a temperature sensor that monitors a temperature differential between the sample and reference cells. The computer program stores in memory information sufficient to determine temperature differentials between the sample and reference cells that arise in response to each change in the pressure applied by the pressure system. For example, the program can store either the actual temperature differentials between the cells or the differential power applied to the sample cell versus the reference cell in order to maintain the sample and reference cells at substantially equal temperatures.
In another aspect, the invention features a calorimeter that includes a sample cell, a reference cell, a pressure system in continuous communication with both the sample cell and the reference cell, and a pressure controller that controls pressure applied by the pressure system. The pressure system is configured to apply a pressure to both cells, and the pressure controller is configured to vary the pressure applied by the pressure system.
Embodiments of this aspect of the invention can further include, e.g., a heat monitoring system that determines the differential heat effect between the sample and reference cells in response to a change in the pressure applied by the pressure system.
In another aspect, the invention features a computer program, disposed on a computer-readable medium, for automating operation of a microcalorimeter that includes features of the calorimeters described above. The computer program includes instructions for causing a processor to periodically vary a pressure applied to the sample and reference cells, and store in memory information sufficient to determine temperature differentials that result when the pressure is varied. The computer program can also cause the heating assembly to scan the temperature of the sample and reference cells at a rate specified by a user.
In another aspect, the invention features a method of performing calorimetry. The method includes: (1) providing a calorimeter that has a reference cell and a sample cell, where the reference cell contains a liquid, and the sample cell contains a solution that includes the liquid and a test substance; (2) varying the pressure above the liquid in the sample cell and the solution in the reference cell; and (3) determining a differential heat effect between the sample cell and the reference cell in response to a change in the pressure applied by the pressure system.
Embodiments of this aspect of the invention may include one or more of the following features. The varying step can include applying a pressure perturbation to both the sample cell and the reference cell. The determining step can include measuring a difference between the temperature of the sample cell and the temperature of the reference cell.
In another aspect, the invention features a method of determining a thermal coefficient of expansion of a substance. The method includes: (1) providing a first liquid holder containing the substance, and a second liquid holder containing a solution in which the substance is not present; (2) applying a pressure perturbation to the solutions in the first and second liquid holders at a known temperature; (3) determining a differential heat effect between the first and second liquid holders in response to the pressure perturbation; (4) calculating, from the differential heat effect, the heat effect of the substance in response to the pressure perturbation; and (5) using the first and second laws of thermodynamics to determine the thermal coefficient of expansion of the substance at the known temperature. The applying step through the using step can then be repeated at a plurality of known temperatures, allowing construction of a function for the thermal coefficient of expansion of the substance as a function of temperature.
In another aspect, the invention features a method of determining the change in volume occupied by a target molecule (e.g., a protein, an oligonucleotide, a lipid, or a carbohydrate) as the molecule transitions from a first structure to a second structure in response to a change in temperature. The method includes: (1) providing a first liquid holder containing a plurality of the target molecules, and a second liquid holder containing no target molecules; (2) applying a pressure perturbation to the contents of the first and second liquid holders at a plurality of different temperatures, where the plurality of temperatures includes temperatures at which the molecules are transitioning from the first structure to the second structure; (3) determining a differential heat effect between the first and second liquid holders in response to each pressure perturbation; (4) calculating, from each differential heat effect, the heat effect of the molecule in response to each pressure perturbation; and (5) calculating the change in volume occupied by the molecule from a data set that includes the pressure perturbation values, the temperatures at which each pressure perturbation was applied, the volume of an individual target molecule in either its first or its second structure, and the heat effect of the molecule for each pressure perturbation.
The second calculating step can include: (a) determining the thermal coefficient of expansion, xcex1, of the molecule at each temperature T in the data set; (b) constructing a function xcex1(T) from the data set; (c) identifying, from the data set, a transition temperature range during which the molecule is actively transitioning from the first structure to the second structure; (d) constructing a second function xcex12(T) for the transition range, where xcex12(T) represents the thermal expansion coefficients that would have been measured in the transition range if the molecule did not change volume as it transitioned from its first to its second structure; (e) calculating the total area between xcex1(T) and xcex12(T) over the transition range; and (f) multiplying the total difference by the molar volume of the molecule.
Embodiments of the invention may include one or more of the following advantages. The new calorimeters allow a user to vary more than one intensive variable, e.g., temperature and pressure, or concentration and pressure, while conducting an experiment. The additional data gathered by applying pressure perturbations while varying temperature or concentration allows calculation of, e.g., the thermal coefficient of expansion of a test substance, and the change in volume occupied by a test substance as it changes structure in solution.
Operation of the new calorimeters can be automated by new software.